DESCRIPTION: This renewal proposal seeks to understand the molecular basis of the regulation of alternative pre-MRNA splicing, especially as it pertains to regulation of gene expression during normal erythroid differentiation. As a paradigm for these analyses, regulation of exon 16 of protein 4.1 pre-MRNA will be explored. Exon 16 encodes a 21 amino acid cassette within the spectrin-actin binding domain that endows protein 4.1 with the capacity to promote association of actin with spectrin in the membrane skeleton. This exon is absent (spliced out) in the mature MRNA of early erythroid progenitors but present (spliced in) in 4.1 MRNA of late progenitors. Dr. Conboy hypothesizes that regulation of exon 16 splicing depends on both cis- regulatory sequence elements in 4.1 pre-MRNA and trans-acting RNA binding protein/splicing factors. To generate an RNA substrate for these mechanistic studies, a simple 3 exon minigene containing exon 16 flanked by native intron sequences and constitutive exons, has been constructed. This model 4.1 pre- MRNA has been shown to participate in the alternative splicing program following by intact 4.1 pre-MRNA, yielding two products differing in inclusion of exon 16. Dr. Conboy proposed to employ this minigene construct to i) characterize cis elements that may positively or negatively modulate exon 16 splicing, ii) test effects of known alternative splicing factors of the SR family on exon 16 splicing, iii) employ RNA affinity chromatography to isolate and clone new trans factors involved in 4.1 pre-MRNA splicing, and iv) characterize the expression of the alternative splicing factors during erythroid development. For analyses of 4.1 pre-MRNA splicing, 3 model splicing systems will be employed: microinjected Xenopus oocytes, nuclear extracts in vitro, and transfected MEL cells. These studies should allow a detailed explanation of the regulation of a critical splicing switch during erythroid development that allows stabilization of the erythrocyte membrane skeleton.